The proposed research will explore the factors which permit multielectron reductases (which transfer 4-6 electrons to bound substrates without release of intermediates: examples are cytochrome oxidase, nitrogenase, sulfite reductase, nitrite reductase) to 1) catalyze facile transfer of electrons more than one at a time to bound substrates while using what appear to be one-electron transferrring groups, and 2) hold tightly to all bound intermediates until transfer of all electrons is complete. We will focus on determination of the structure and mechanisms of action of sulfite and nitrite reductases, both of which contain relatively simple active centers: an Fe4S4 cluster chemically linked to a novel Fe-tetrahydroporphyrin termed siroheme, on a polypeptide chain of ca. 60,000 molecular weight (monomeric in solution). Both enzymes catalyze 6-electron reductions of SO32-to H2S and NO2- to NH3. We will study the structure of E. coli sulfite reductase by X-ray crystallography, and use a variety of spectroscopic techniques (optical, EPR, Mossbauer, ENDOR, as well as several others), magnetic susceptibility, and rapid kinetic approaches to explore the chemical structure of both sulfite and nitrite reductases, their complexes with substrates and competitive inhibitors, and of intermediates in the multielectron reduction process. We will also explore properties of siroheme itself which may particularly suit it for catalysis of sulfite and nitrite reduction. We are particularly interested in determining whether substrates and intermediates are bound simultaneously to both metal prosthetic groups during catalysis and whether unusual oxidation states of these prosthetic groups are involved (thus facilitating substrate/intermediate binding and minimizing the number of intermediates necessary in the 6-electron transfer reaction). These questions are of central importance to our understanding of the function of all multielectron reductase reactions.